Synthesis and Study of Some New Quinolone Derivatives Containing a 3-acetyl Coumarin for Their Antibacterial and Antifungal Activities.

A series of N-[2-(8-metoxy-2H-chromen-2-one)ethyl] piperazinyl quinolones containing a carbonyl related functional groups (oxo or oxyimino) on the ethyl spacer of coumarin and piperazin rings was synthesized and studied for their antibacterial and antifungal activities . The synthesis of compounds (6a-6l) was achieved through the versatile and efficient synthetic route that involved reaction of quinolones with appropriately α- bromo ketone or α- bromo oxime derivatives (2, 2a-c). The structures of the new compounds were confirmed by IR, Mass, 1H-NMR and 13C-NMR spectra. More good activities against gram-positive and gram-negative are shown in all compounds. The antifungal data reveals that all compounds have shown weak antifungal activity as compared to Nistatin.


Introduction
Quinoline derivatives show the main grade of heterocycles, and a number of provisions have been identified since the late 1800s. The quinoline ring system takes place in different natural products, especially in alkaloids. Derivatives of quinolones have been clinically successful and are used to care bacterial infections in either community or hospital environments. Since then, quinolones were the only synthetic agents that play a main role in the treatment of bacterial community or hospital needed illnesses. Quinolones aim bacterial type II topoisomerases, gen erally DNA gyrase in Gram-negative bacteria and DNA topoisomerase IV in Gram-positive bacteria. Quinolines are also identified for their formation of conjugated molecules and polymers that blend improved electronic, optoelectronic, or nonlinear optical features with great mechanical properties (1)(2)(3)(4)(5)(6).
Piperazine, aminopyrrolidine, and their replaced derivatives have been the most successfully applied side chains, as proved by the compounds recently on the market. The biological activity of quinoline compounds has been found to have antiasthmatic, antibacterial, anti-inflammatory, and antihypertensive properties. The quinoline shape is often used for the design of many synthetic compounds with diverse pharmacological properties. The epidemic use of these compounds and the need for clinical and pharmacological survey require fast and sensitive analytical ways for identification of its presence in biological liquid (7)(8)(9)(10)(11)(12).
In fact, the newer fluoro quinolones raise with the development of 7-piperazinyl quinolones, such as ciprofloxacin 1, norfloxacin 2, enoxacin 3 and levofloxacin 4 ( Figure 1). The most extreme structural difference has been done on amines at the 7-position, partially due to the ease of their introduction through a nucleo philic aromaticsubstitution reaction to the related halide. We have focused our attention on modification of the C-7 basic group of the quinolone (13-18).
The coumarins inhibit ATPase activity of DNA gyrase by competing with ATP for binding to the B subunit of the enzyme. However, due to their toxicity in eukaryotes, their poor wate rsolubility, and their low activity against gramnegative bacteria, no pharmaceutically useful drug has, so far, been derived from the coumarins (19). However, renewed interest in coumarin antibiotics came from their potent gram-positive and gram-negative antibacterial activity and, which are currentlyone of the major concerns in treatment of bacterial infections (20).
In continuing our efforts to find new quinolone, we target to combine the structural characteristics of our promising antibacterial N-(2-arylethyl) piperazinyl quinolones and coumarin antibacterial drug. Therefore here, we like to state the synthesis and antibacterial activity of N-[2-(coumarin-3-yl) ethyl] piperazinyl quinolones 6 (schme 1).

Materials and methods Chemistry
Chemical reagents and all solvents used in this research were bought from Merck AG (Darmstadt, Germany). Melting points were determined in open glass capillaries using Bibby Stuart Scientific SMP 3 apparatus (Bibby Sterlin Ltd, U.K.) and are uncorrected. The FT-IR spectra were obtained on a Shimadzu 470 spectrophotometer (potassium bromide disks; Shimadzu, Tokyo, Japan). Mass spectra were also recorded with an Agilent Technologies 5973, Mass Selective Detector (MSD) spectrometer (Wilmigton, USA). 1 H-NMR spectra were recorded using a Bruker 500 spectrometer and 13 C-NMR spectra were recorded using a Bruker 300 spectrometer (Bruker Bioscience, Billerica, MA, USA), and chemical shifts are expressed as δ (ppm) with tetramethylsilane as internal standard. Merck silica gel 60 F 254 plates were used for analytical TLC (Merck).

Antimicrobial and Antifungal Assay
The antimicrobial activity was assayed by cup-plate agar diffusion method (24-25) by measuring inhibition zones in mm. In vitro antimicrobial activity of all synthesized compounds and standard drugs have been evaluated against two strains of bacteria which include gram-positive bacteria such as, Bacillus subtilis PTCC 1207 and gram-negative bacteria such as Escherichia coli PTCC 1047 and fungus Candida kefyr ATCC 38296. The antibacterial and antifungal activity was compared with standard drugs.
The purified products were screened for their antibacterial activity by using cup-plate agar diffusion method. The nutrient agar broth prepared by the usual method, was inoculated aseptically with 0.5 mL of 24 h old subculture of, Bacillus subtilis PTCC 1047, and Escherichia coli PTCC 1047 in separate conical flasks at 40-50 º C and mixed well by gently shaking. About 25 mL of the contents of the flask was poured and evenly spread in a petri dish (90 mm in diameter) and allowed to set for 2 h. The cups (10 mm in diameter) were formed by the help of borer in agar medium and filled with 0.4 mL (400μg / mL) solution of sample in DMSO.
The plates were incubated at 37 º C for 24 h and the control was also maintained with 0.4 mL of DMSO in a similar manner and the zones of inhibition of the bacterial growth were measured in millimeter and recorded in (Table 2 and Figure  2). Candida kefyr ATCC 38296 was employed for testing antifungal activity by cup-plate agar diffusion method. The culture was maintained on Sub rouse dextrose agar slants. Sterilized Sub rouse dextrose agar medium was inoculated with 72 h old 0.5 mL suspension of fungal spores in a separate flask. About 25 mL of the inoculated medium was evenly spread on a sterilized petri dish and allowed to set for 2 h. The cups (10 mm in diameter) were punched in a petri dish and loaded with 0.4 mL (400 μg/ mL) of solution of sample in DMSO. The plates were incubated at 30 º C for 48 h. After the completion of incubation period, the zones of inhibition of growth in the form of diameter in mm were measured. Along with the test solution in each petri dish one cup was filled up with solvent which acts as a control. The zones of inhibition are recorded in (Table 2 and Figure 2).

Results and Discussion
In this study the structure of the synthesized compounds was elucidated by means of IR, 1 H-NMR, 13 C-NMR and Mass. All the compounds were evaluated for antibacterial and antifungal activities by cup-plate method. The antimicrobial activity of tested compounds against different strains of bacteria and fungus is shown in (Table 2 and Figure 2). The newly synthesized compounds 6a-l were evaluated for their in-vitro antibacterial activity against Bacillus subtilis PTCC 1207, Escherichia coli PTCC 1047 and Candida kefyr ATCC 38296 using conventional by cup-plate agar diffusion method (24). The zone of growth inhibition values was determined by comparison to standard drugs. The zone of growth inhibition obtained for compounds 6a-l are presented in (Table 2 and Figure 2).
The zone of growth inhibition values of the test derivatives indicated that most compounds exhibit good activity against gram-positive and gram-negative bacteria. Antibacterial screening of compounds 6a-l against gram-positive and gram-negative bacteria reveals that compounds 6g, 6h and 6i exhibit the most potent in-vitro antibacterial activity. All compounds show improvement of activity against gram-positive bacteria in comparison to standard drugs.
Generally, in both gram-positive and gramnegative bacteria, better results are obtained with cyclopropyl and ethyl at the N-1position of the quinolone ring and 2-NOCH 3 on the ethyl spacer of coumarin and piperazine rings.
Conclusion some of the new N-[2-(coumarin-3-yl) ethyl]piperazinyl quinolones 6 containing a carbonyl related functional groups (ketone, oxime, O-methyloxime, and O-benzyloxime) on the ethyl spacer showed considerable antibacterial activity and modification of the position 8 and N-1 substituent on the quinolone ring, and ethyl spacer functionality produced relatively major changes in terms of activity. In general, the results of antibacterial evaluation of the test compounds in comparison with the reference drugs indicated that compounds6g, 6 h and 6i showed comparable or more potent antibacterial activity with respect to the reference drugs against all tested species. The antifungal data reveals that all compounds have shown weak antifungal activity as compared to Nistatin.

Acknowledgment
The authors of the present paper acknowledge